Creation of AlkB Mutants for Protection of Bone Marrow

描述(申请人提供)：甲基化药物是治疗癌症的主要药物，也是少数几种可用于治疗脑瘤的药物之一。 负责维持血细胞供应的细胞，即“造血祖细胞”，对这些药物高度敏感，这会导致严重的副作用，包括出血和感染。 抗性基因可以保护造血祖细胞免受甲基化的毒性影响。最有效的是AGT，它直接去除细胞遗传物质(DNA)甲基化引起的有毒修饰。然而，AGT不识别某些有毒的DNA修饰。在这里，我建议使用另一种基因(AlkB)，它针对这些替代损伤中的一些，以提高对甲基化毒性影响的保护。这种基因最近才被鉴定，因此应该会增强AGT的保护作用。此外，与AGT不同的是，AlkB在介导修复反应后仍然具有活性，这意味着AlkB修复应该更易于调节。 在这里，我提出了一种强大的方法来识别人类AlkB同源物(ABH2)的变体，并改善了修复性能。具体地说，我想增强这种酶识别临床上使用的试剂产生的损伤的能力。选定的变种将在培养的人类祖细胞和小鼠动物模型中进行测试，以防止高剂量这些药物的治疗。最终，这项工作中发现的突变应该会改善甲基化药物副作用的管理，并允许使用更高剂量的药物进行更有效的治疗。

DESCRIPTION (provided by applicant): Methylating agents are mainstay drugs for the treatment of cancer and are amongst the few agents available against brain tumors. Cells responsible for keeping the blood cell supply, "blood progenitor cells", are highly sensitive to these agents, which results in severe side-effects, including bleeding and infection. Blood progenitor cells can be protected from the toxic effects of methylation by resistance genes. The most effective one is AGT, which directly removes toxic modifications caused by methylation of the cell's genetic material (DNA). However, AGT does not recognize certain toxic DNA modifications. Here I propose the use of a different gene (AlkB), which targets some of these alternative lesions, to improve protection against the toxic effects of methylation. This gene, which has only recently been characterized, should therefore enhance AGT protection. Moreover, unlike AGT, AlkB remains active after mediating a repair reaction, which implies that AlkB repair should be more amenable to modulation. Here I propose a powerful approach to identify variants of one human homologue of AlkB (ABH2) with improved repair performance. Specifically, I want to enhance the enzyme's ability to recognize lesions generated by agents used in the clinic. Selected variants will be tested in human progenitor cells in culture and in a mouse animal model for protection against treatment with high doses of these agents. Ultimately, the mutants identified in this work should improve the management of side effects of methylating agents and would also allow the use of higher doses for more effective treatment.